The Golgi consists of a stack of flattened membrane compartments that is essential for the sequential processing and sorting of all secretory and transmembrane proteins before they are delivered to their final destinations. Yet a clear understanding of the role and regulation of Golgi stacking on the protein trafficking functions of the Golgi has remained elusive due to the lack of experimental methods to induce Golgi unstacking in vivo. For example, many studies have relied on investigating the disassembled Golgi from mitotic extracts that is limited to in vitro experiments. RNAi-mediated knockdown of potential stacking factors did not produce unstacked Golgi, perhaps due to compensatory mechanisms that kick-in during the long silencing period. The long-term objective of this project is to investigate the structure-function relationship of the Golgi by devising a new approach to rapidly unstack the Golgi in live cells. Then, its effect on Golgi function and dynamics will be directly visualized in real time using live cell imaging. The first aim will involve the constructon of a knock-sideways system for Golgi stacking factors GRASP65 and GRASP55. This will allow their drug-induced inactivation at the Golgi in cells where RNAi has silenced the endogenous counterpart. The unstacking of nocodazole-induced peripheral Golgi mini-stacks will be monitored by triple color total internal reflection fluorescence (TIRF) microscopy, which has the resolution needed to distinguish cis- and trans-cisternae. The second aim will set up a live cell imaging assay that can visualize intra-Golgi trafficking in real time. TIRF microscopy will be used to directly monitor cargo entry into the cis- or trans-cisternae in stacked and unstacked Golgi in order to test the hypothesis that Golgi stacking is mandatory for intra-Golgi trafficking. This work will provide important insight into the most conserved feature of Golgi membrane structure and function. In addition, a better understanding of the mechanism of Golgi misregulation in human genetic disorders and cancers related to membrane trafficking may be attained. PUBLIC HEALTH RELEVANCE: Misregulation of the membrane trafficking pathway in the Golgi, by which cargo proteins are sequentially processed and sorted before delivery to their site of function, is linked to multiple human genetic disorders and cancers. Despite the continuous membrane flux, the Golgi maintains a stacked organization of membrane compartments that is central to its processing functions. Yet it is unclear how this unique organization regulates membrane trafficking due to lack of amenable methods for unstacking the Golgi. A novel approach for rapid Golgi unstacking in vivo will be developed to allow investigation of its effect on intra-Golgi trafficking by super- resolution live cell imaging. This work will provide the mechanistic basis for many human diseases caused by mutations in Golgi membrane trafficking proteins, and may guide therapeutic strategies that target misregulated Golgi in cancer.